siRNA phospholipid conjugate

ABSTRACT

siRNA-conjugated liposomes and micelles, methods of making such conjugates, and methods of using such conjugates, such as for the delivery of siRNA to cells to reduce expression of target polypeptides in such cells, are described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Stage Entry of PCT/US10/41975, filed Jul.14, 2010, which claims the benefit of U.S. Provisional Application No.61/225,298, filed Jul. 14, 2009, the entire contents of which are herebyincorporated by reference herein.

FIELD OF THE INVENTION

This disclosure is in the fields of molecular biology and medicine. Morespecifically, this disclosure relates to siRNA control of geneexpression.

BACKGROUND

Small interfering RNA (siRNA) is a short double stranded RNA. It behaveslike a mediator of the RNA interference phenomenon silencing thespecific gene expression by triggering the cleavage of a targetmessenger RNA (mRNA) at post-transcriptional level in the cytoplasm(Hamilton, et al., Science 1999; Elbashir, et al., Nature (2001)). siRNAis a powerful tool to control cellular processes at apost-transcriptional level. Its great potency is due to the highsequence-specific inhibition efficiency (Novina et al., Nature (2004)430:161-164). Thus, siRNA strategy has been strongly considered for thedown-regulation of certain proteins in the areas of functional genomicsand genomic therapeutics.

Although siRNA has been used as a therapeutic agent for various geneticdiseases, its therapeutic application is still limited because of itsinstability against nucleases. Thus, what is needed are siRNA moleculesthat are more stable, and thus more active and useful as therapeuticagents.

SUMMARY

The invention is based, in part, on the discovery that siRNA can bereversibly conjugated to a phospholipid, such as a phospholipid within aliposome or a micelle, and the siRNA can be unconjugated upon exposureto reducing conditions, such as inside a cell.

Accordingly, in one aspect, the disclosure features a conjugated siRNAcomposition comprising a micelle or a liposome, the micelle or theliposome comprising phospholipids; and an siRNA reversibly conjugated toa first phospholipid of the micelle or the liposome.

In some embodiments, the siRNA is reversibly conjugated to the firstphospholipid by a disulfide bond. In certain embodiments, the siRNA isunconjugated from the first phospholipid upon exposure to reducingconditions.

In certain embodiments, the siRNA reversibly conjugated to the firstphospholipid has increased stability relative to the same siRNA notconjugated to the first phospholipid. In particular embodiments, thesiRNA reversibly conjugated to the first phospholipid is about 10% toabout 10000%, about 20% to about 1000%, about 30% to about 500%, about40% to about 400%, about 50% to about 300%, about 60% to about 200%,about 70% to about 150%, about 80% to about 100%, about 25%, about 50%,about 75%, about 100%, about 150%, about 200%, about 250%, about 500%,about 1000%, about 1500%, about 2000%, about 2500%, about 5000%, about10000%, about 50000%, or more, more stable than the same siRNA notconjugated to the first phospholipid.

In some embodiments, the siRNA reversibly conjugated to the firstphospholipid exhibits reduced degradation by RNase relative to the samesiRNA not conjugated to the first phospholipid. In particularembodiments, the level of degradation by RNase is reduced by about 10%,by about 20%, by about 30%, by about 40%, by about 50%, by about 60%, byabout 70%, by about 80%, by about 90%, by about 95%, or by about 100%,relative to the same siRNA not conjugated to the first phospholipid.

In certain embodiments, the siRNA reversibly conjugated to the firstphospholipid exhibits an increased half-life when administered into asubject, relative to the same siRNA not conjugated to the firstphospholipid. In particular embodiments, the increase in half-life ofthe siRNA reversibly conjugated to the first phospholipid is about 10%to about 10000%, about 20% to about 1000%, about 30% to about 500%,about 40% to about 400%, about 50% to about 300%, about 60% to about200%, about 70% to about 150%, about 80% to about 100%, about 25%, about50%, about 75%, about 100%, about 150%, about 200%, about 250%, about500%, about 1000%, about 1500%, about 2000%, about 2500%, about 5000%,about 10000%, about 50000%, or more, relative to the same siRNA notconjugated to the first phospholipid. In particular embodiments, thehalf-life of the siRNA reversibly conjugated to the first phospholipidexhibits a half-life, when administered into a subject, of about 6hours, about 12 hours, about 24 hours, about 2 days, about 3 days, about4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9days, about 10 days, about 11 days, about 12 days, about 13 days, about14 days, or longer.

In some embodiments, about 1% to about 100% of the phospholipids of theliposome or the micelle are reversibly conjugated to the siRNA. In otherembodiments, about 1%, about 5%, about 10%, about 20%, about 30%, about40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about100% of the phospholipids of the liposome or the micelle are reversiblyconjugated to the siRNA.

In some embodiments, the siRNA comprises about 0.5% to about 90% of theliposome or the micelle by weight. In particular embodiments, the siRNAcomprises about 0.5%, about 1%, about 5%, about 10%, about 20%, about30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%of the liposome or micelle by weight.

In some embodiments, the micelle or the liposome further comprisespolyethylene glycol (PEG) conjugated to a second phospholipid of themicelle or the liposome. In certain embodiments, the first phospholipidand the second phospholipid are different. In other embodiments, thefirst phospholipid and the second phospholipid are the same. In someembodiments, the first phospholipid is phosphatidylcholine (PC),phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid(PA), phosphatidyethanolamine (PE), phosphatidylserine (PS), orphosphoethanolamine.

In some embodiments, about 50% of the phospholipids are reversiblyconjugated to the siRNA and about 50% of the phospholipids areconjugated to PEG, about 40% of the phospholipids are reversiblyconjugated to the siRNA and about 60% of the phospholipids areconjugated to PEG, about 30% of the phospholipids are reversiblyconjugated to the siRNA and about 70% of the phospholipids areconjugated to PEG, about 20% of the phospholipids are reversiblyconjugated to the siRNA and about 80% of the phospholipids areconjugated to PEG, about 10% of the phospholipids are reversiblyconjugated to the siRNA and about 90% of the phospholipids areconjugated to PEG, about 60% of the phospholipids are reversiblyconjugated to the siRNA and about 40% of the phospholipids areconjugated to PEG, about 70% of the phospholipids are reversiblyconjugated to the siRNA and about 30% of the phospholipids areconjugated to PEG, about 80% of the phospholipids are reversiblyconjugated to the siRNA and about 20% of the phospholipids areconjugated to PEG, or about 90% of the phospholipids are reversiblyconjugated to the siRNA and about 10% of the phospholipids areconjugated to PEG.

In certain embodiments, the micelle or the liposome comprises a wt/wtratio of (phospholipids reversibly conjugated to the siRNA) to(phospholipids conjugated to PEG) of about 1:10 to about 1:5000, about1:50 to about 1:2500, about 1:100 to about 1:2000, about 1:150 to about1:1500, about 1:200 to about 1:1000; about 1:250 to about 1:900, about1:300 to about 1:800, about 1:400 to about 1:750, or about 1:500 toabout 600.

In some embodiments, the composition further comprises a targeting agentconjugated to a phospholipid of the micelle or liposome. In someembodiments, the targeting agent is conjugated to the firstphospholipid. In other embodiments, the targeting agent is conjugated tothe second phospholipid. In yet other embodiments, the targeting agentis conjugated to both the first and the second phospholipid. In stillother embodiments, the targeting agent is conjugated to a phospholipidnot conjugated to the siRNA or the PEG.

In another aspect, the disclosure features an siRNA compositioncomprising an siRNA reversibly conjugated to a phospholipid. In someembodiments, the siRNA is reversibly conjugated to the phospholipid by adisulfide bond. In certain embodiments, the siRNA is unconjugated fromthe phospholipid upon exposure to reducing conditions.

In certain embodiments, the phospholipid is phosphatidylcholine (PC),phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid(PA), phosphatidyethanolamine (PE), phosphatidylserine (PS), orphosphoethanolamine.

In particular embodiments, the siRNA reversibly conjugated to thephospholipid has increased stability relative to the same siRNA notconjugated to the phospholipid. In certain embodiments, the siRNAreversibly conjugated to the phospholipid is about 10% to about 10000%,about 20% to about 1000%, about 30% to about 500%, about 40% to about400%, about 50% to about 300%, about 60% to about 200%, about 70% toabout 150%, about 80% to about 100%, about 25%, about 50%, about 75%,about 100%, about 150%, about 200%, about 250%, about 500%, about 1000%,about 1500%, about 2000%, about 2500%, about 5000%, about 10000%, about50000%, or more, more stable than the same siRNA not conjugated to thephospholipid.

In some embodiments, the siRNA reversibly conjugated to the phospholipidexhibits reduced degradation by RNase relative to the same siRNA notconjugated to the phospholipid. In particular embodiments, the level ofdegradation by RNase is reduced by about 10%, by about 20%, by about30%, by about 40%, by about 50%, by about 60%, by about 70%, by about80%, by about 90%, by about 95%, or by about 100%, relative to the samesiRNA not conjugated to the phospholipid.

In certain embodiments, the siRNA reversibly conjugated to thephospholipid exhibits an increased half-life when administered into asubject, relative to the same siRNA not conjugated to the phospholipid.In particular embodiments, the increase in half-life of the siRNAreversibly conjugated to the phospholipid is about 10% to about 10000%,about 20% to about 1000%, about 30% to about 500%, about 40% to about400%, about 50% to about 300%, about 60% to about 200%, about 70% toabout 150%, about 80% to about 100%, about 25%, about 50%, about 75%,about 100%, about 150%, about 200%, about 250%, about 500%, about 1000%,about 1500%, about 2000%, about 2500%, about 5000%, about 10000%, about50000%, or more, relative to the same siRNA not conjugated to thephospholipid. In particular embodiments, the half-life of the siRNAreversibly conjugated to the phospholipid exhibits a half-life, whenadministered into a subject, of about 6 hours, about 12 hours, about 24hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6days, about 7 days, about 8 days, about 9 days, about 10 days, about 11days, about 12 days, about 13 days, about 14 days, or longer.

In another aspect, the disclosure features a method of inhibitingexpression of a target polypeptide in a subject, the method comprisingadministering to the subject a conjugated siRNA composition in an amountsufficient to inhibit expression of the target polypeptide in a cell ofthe subject, wherein the conjugated siRNA composition comprises (i) amicelle or a liposome, the micelle or the liposome comprisingphospholipids; and (ii) an siRNA reversibly conjugated to a firstphospholipid of the micelle or the liposome; and allowing the siRNA tounconjugate from the first phospholipid of the micelle or the liposomewithin the cell of the subject, thereby inhibiting the expression of thetarget polypeptide in the subject.

In some embodiments, the siRNA is reversibly conjugated to the firstphospholipid by a disulfide bond. In certain embodiments, the siRNA isunconjugated from the first phospholipid upon exposure to reducingconditions within the cell.

In particular embodiments, the expression of the target polypeptide isreduced by about 10%, about 20%, about 30%, about 40%, about 50%, about60%, about 70%, about 80%, about 90%, or about 100%, relative to theexpression of the target polypeptide in the absence of administration ofthe conjugated siRNA composition.

In certain embodiments, the siRNA reversibly conjugated to the firstphospholipid has increased stability relative to the same siRNA notconjugated to the first phospholipid. In some embodiments, the siRNAreversibly conjugated to the first phospholipid is about 10% to about10000%, about 20% to about 1000%, about 30% to about 500%, about 40% toabout 400%, about 50% to about 300%, about 60% to about 200%, about 70%to about 150%, about 80% to about 100%, about 25%, about 50%, about 75%,about 100%, about 150%, about 200%, about 250%, about 500%, about 1000%,about 1500%, about 2000%, about 2500%, about 5000%, about 10000%, about50000%, or more, more stable than the same siRNA not conjugated to thefirst phospholipid.

In some embodiments, the siRNA reversibly conjugated to the firstphospholipid exhibits reduced degradation by RNase relative to the samesiRNA not conjugated to the first phospholipid. In particularembodiments, the level of degradation by RNase is reduced by about 10%,by about 20%, by about 30%, by about 40%, by about 50%, by about 60%, byabout 70%, by about 80%, by about 90%, by about 95%, or by about 100%,relative to the same siRNA not conjugated to the first phospholipid.

In certain cases, the siRNA reversibly conjugated to the firstphospholipid may exhibit an increased half-life when administered to thesubject, relative to the same siRNA not conjugated to the firstphospholipid. In some embodiments, the increase in half-life of thesiRNA reversibly conjugated to the first phospholipid is about 10% toabout 10000%, about 20% to about 1000%, about 30% to about 500%, about40% to about 400%, about 50% to about 300%, about 60% to about 200%,about 70% to about 150%, about 80% to about 100%, about 25%, about 50%,about 75%, about 100%, about 150%, about 200%, about 250%, about 500%,about 1000%, about 1500%, about 2000%, about 2500%, about 5000%, about10000%, about 50000%, or more, relative to the same siRNA not conjugatedto the first phospholipid. In particular embodiments, the half-life ofthe siRNA reversibly conjugated to the first phospholipid exhibits ahalf-life, when administered into the subject, of about 6 hours, about12 hours, about 24 hours, about 2 days, about 3 days, about 4 days,about 5 days, about 6 days, about 7 days, about 8 days, about 9 days,about 10 days, about 11 days, about 12 days, about 13 days, about 14days, or longer.

In some embodiments, about 1% to about 100% of the phospholipids of theliposome or the micelle are reversibly conjugated to the siRNA. In otherembodiments, about 1%, about 5%, about 10%, about 20%, about 30%, about40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about100% of the phospholipids of the liposome or the micelle are reversiblyconjugated to the siRNA.

In certain embodiments, the siRNA comprises about 0.5% to about 90% ofthe liposome or the micelle by weight. In particular embodiments, thesiRNA comprises about 0.5%, about 1%, about 5%, about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, orabout 90% of the liposome or micelle by weight.

In some embodiments, the micelle or the liposome further comprisespolyethylene glycol (PEG) conjugated to a second phospholipid of themicelle or the liposome. In certain embodiments, the first phospholipidand the second phospholipid are different. In other embodiments, thefirst phospholipid and the second phospholipid are the same. In someembodiments, the first phospholipid is phosphatidylcholine (PC),phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid(PA), phosphatidyethanolamine (PE), phosphatidylserine (PS), orphosphoethanolamine.

In some embodiments, about 50% of the phospholipids are reversiblyconjugated to the siRNA and about 50% of the phospholipids areconjugated to PEG, about 40% of the phospholipids are reversiblyconjugated to the siRNA and about 60% of the phospholipids areconjugated to PEG, about 30% of the phospholipids are reversiblyconjugated to the siRNA and about 70% of the phospholipids areconjugated to PEG, about 20% of the phospholipids are reversiblyconjugated to the siRNA and about 80% of the phospholipids areconjugated to PEG, about 10% of the phospholipids are reversiblyconjugated to the siRNA and about 90% of the phospholipids areconjugated to PEG, about 60% of the phospholipids are reversiblyconjugated to the siRNA and about 40% of the phospholipids areconjugated to PEG, about 70% of the phospholipids are reversiblyconjugated to the siRNA and about 30% of the phospholipids areconjugated to PEG, about 80% of the phospholipids are reversiblyconjugated to the siRNA and about 20% of the phospholipids areconjugated to PEG, or about 90% of the phospholipids are reversiblyconjugated to the siRNA and about 10% of the phospholipids areconjugated to PEG.

In particular embodiments, the micelle or the liposome comprises a wt/wtratio of (phospholipids reversibly conjugated to the siRNA) to(phospholipids conjugated to PEG) of about 1:10 to about 1:5000, about1:50 to about 1:2500, about 1:100 to about 1:2000, about 1:150 to about1:1500, about 1:200 to about 1:1000; about 1:250 to about 1:900, about1:300 to about 1:800, about 1:400 to about 1:750, or about 1:500 toabout 600.

In certain embodiments, the conjugated siRNA composition furthercomprises a targeting agent conjugated to a phospholipid of the micelleor the liposome. In some embodiments, the targeting agent mediatesuptake of the conjugated siRNA composition by the cell. In someembodiments, the targeting agent is conjugated to the firstphospholipid. In other embodiments, the targeting agent is conjugated tothe second phospholipid. In yet other embodiments, the targeting agentis conjugated to both the first and the second phospholipid. In yetother embodiments, the targeting agent is conjugated to a phospholipidnot conjugated to the siRNA or the PEG.

In certain embodiments, the subject is a human, ape, monkey, orangutan,chimpanzee, dog, cat, guinea pig, rabbit, rat, mouse, horse, cattle, orcow.

In another aspect, the disclosure features a method of inhibitingexpression of a target polypeptide in a cell, the method comprisingdelivering into a cell a conjugated siRNA composition in an amountsufficient to inhibit expression of the target polypeptide in the cell,wherein the conjugated siRNA composition comprises (i) a micelle or aliposome, the micelle or the liposome comprising phospholipids; and (ii)an siRNA reversibly conjugated to a first phospholipid of the micelle orthe liposome; and allowing the siRNA to unconjugate from the firstphospholipid of the micelle or the liposome within the cell, therebyinhibiting the expression of the target polypeptide in the cell.

In some embodiments, the siRNA is reversibly conjugated to the firstphospholipid by a disulfide bond. In certain embodiments, the siRNA isunconjugated from the first phospholipid upon exposure to reducingconditions within the cell.

In particular embodiments, the expression of the target polypeptide isreduced by about 10%, about 20%, about 30%, about 40%, about 50%, about60%, about 70%, about 80%, about 90%, or about 100%, relative to theexpression of the target polypeptide in the absence of administration ofthe conjugated siRNA composition.

In some embodiments, the siRNA reversibly conjugated to the firstphospholipid has increased stability relative to the same siRNA notconjugated to the first phospholipid. In certain embodiments, the siRNAreversibly conjugated to the first phospholipid is about 10% to about10000%, about 20% to about 1000%, about 30% to about 500%, about 40% toabout 400%, about 50% to about 300%, about 60% to about 200%, about 70%to about 150%, about 80% to about 100%, about 25%, about 50%, about 75%,about 100%, about 150%, about 200%, about 250%, about 500%, about 1000%,about 1500%, about 2000%, about 2500%, about 5000%, about 10000%, about50000%, or more, more stable than the same siRNA not conjugated to thefirst phospholipid.

In certain embodiments, the siRNA reversibly conjugated to the firstphospholipid exhibits reduced degradation by RNase relative to the samesiRNA not conjugated to the first phospholipid. In particularembodiments, the level of degradation by RNase is reduced by about 10%,by about 20%, by about 30%, by about 40%, by about 50%, by about 60%, byabout 70%, by about 80%, by about 90%, by about 95%, or by about 100%,relative to the same siRNA not conjugated to the first phospholipid.

In some embodiments, the siRNA reversibly conjugated to the firstphospholipid exhibits an increased half-life when administered to asubject, relative to the same siRNA not conjugated to the firstphospholipid. In certain embodiments, the increase in half-life of thesiRNA reversibly conjugated to the first phospholipid is about 10% toabout 10000%, about 20% to about 1000%, about 30% to about 500%, about40% to about 400%, about 50% to about 300%, about 60% to about 200%,about 70% to about 150%, about 80% to about 100%, about 25%, about 50%,about 75%, about 100%, about 150%, about 200%, about 250%, about 500%,about 1000%, about 1500%, about 2000%, about 2500%, about 5000%, about10000%, about 50000%, or more, relative to the same siRNA not conjugatedto the first phospholipid. In particular embodiments, the half-life ofthe siRNA reversibly conjugated to the first phospholipid exhibits ahalf-life, when administered into a subject, of about 6 hours, about 12hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5days, about 6 days, about 7 days, about 8 days, about 9 days, about 10days, about 11 days, about 12 days, about 13 days, about 14 days, orlonger.

In certain embodiments, about 1% to about 100% of the phospholipids ofthe liposome or the micelle are reversibly conjugated to the siRNA. Inother embodiments, about 1%, about 5%, about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, orabout 100% of the phospholipids of the liposome or the micelle arereversibly conjugated to the siRNA.

In some embodiments, the siRNA comprises about 0.5% to about 90% of theliposome or the micelle by weight. In particular embodiments, the siRNAcomprises about 0.5%, about 1%, about 5%, about 10%, about 20%, about30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%of the liposome or micelle by weight.

In particular embodiments, the micelle or the liposome further comprisespolyethylene glycol (PEG) conjugated to a second phospholipid of themicelle or the liposome. In certain embodiments, the first phospholipidand the second phospholipid are different. In other embodiments, thefirst phospholipid and the second phospholipid are the same. In yetother embodiments, the first phospholipid is phosphatidylcholine (PC),phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid(PA), phosphatidyethanolamine (PE), phosphatidylserine (PS), orphosphoethanolamine.

In some embodiments, about 50% of the phospholipids are reversiblyconjugated to the siRNA and about 50% of the phospholipids areconjugated to PEG, about 40% of the phospholipids are reversiblyconjugated to the siRNA and about 60% of the phospholipids areconjugated to PEG, about 30% of the phospholipids are reversiblyconjugated to the siRNA and about 70% of the phospholipids areconjugated to PEG, about 20% of the phospholipids are reversiblyconjugated to the siRNA and about 80% of the phospholipids areconjugated to PEG, about 10% of the phospholipids are reversiblyconjugated to the siRNA and about 90% of the phospholipids areconjugated to PEG, about 60% of the phospholipids are reversiblyconjugated to the siRNA and about 40% of the phospholipids areconjugated to PEG, about 70% of the phospholipids are reversiblyconjugated to the siRNA and about 30% of the phospholipids areconjugated to PEG, about 80% of the phospholipids are reversiblyconjugated to the siRNA and about 20% of the phospholipids areconjugated to PEG, or about 90% of the phospholipids are reversiblyconjugated to the siRNA and about 10% of the phospholipids areconjugated to PEG.

In certain embodiments, the micelle or the liposome comprises a wt/wtratio of (phospholipids reversibly conjugated to the siRNA) to(phospholipids conjugated to PEG) of about 1:10 to about 1:5000, about1:50 to about 1:2500, about 1:100 to about 1:2000, about 1:150 to about1:1500, about 1:200 to about 1:1000; about 1:250 to about 1:900, about1:300 to about 1:800, about 1:400 to about 1:750, or about 1:500 toabout 600.

In particular embodiments, the conjugated siRNA composition furthercomprises a targeting agent conjugated to a phospholipid of the micelleor the liposome. In some embodiments, the targeting agent mediatesuptake of the conjugated siRNA composition by the cell. In certainembodiments, the targeting agent is conjugated to the firstphospholipid. In other embodiments, the targeting agent is conjugated tothe second phospholipid. In yet other embodiments, the targeting agentis conjugated to both the first and the second phospholipid. In otherembodiments, the targeting agent is conjugated to a phospholipid notconjugated to the siRNA or the PEG. In some embodiments, the targetingagent mediates delivery into the cell.

In certain embodiments, the cell is within a subject. In someembodiments, the subject is a human, ape, monkey, orangutan, chimpanzee,dog, cat, guinea pig, rabbit, rat, mouse, horse, cattle, or cow.

The following figures are presented for the purpose of illustrationonly, and are not intended to be limiting.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a schematic representation of the synthesis of siRNA-S-S-PE.

FIG. 1B is a photographic representation of the cleavage of disulfidebonds in a 10 mM glutathione solution as monitored by TLC, where “C”represents siRNA-S-S-PE before hydrolysis; “H” represents siRNA afterthe hydrolysis of siRNA-conjugate; and “P” represents free phospholipid.

FIG. 2A is a schematic representation of a siRNA-S-S-PE/PEG-PE mixedmicelle.

FIG. 2B is a graphic representation of mean size of mixed micelleformulations.

FIG. 2C is a representation of a transmission electron micrographshowing the morphology and size distribution of 1:750 (wt/wt)siRNA-PE/PEG-PE micelles.

FIG. 2D is a graphic representation of the mean zeta potential value ofsiRNA-S-S-PE/PEG-PE mixed micelle formulations and plain PEG-PEmicelles.

FIG. 3 is a series of graphic representations of HPLC absorbance versusretention times for siRNA-S-S-PE conjugate micelles (FIG. 3A), PEG-PEmicelles (FIG. 3B), and siRNA-S-S-PE conjugate micelles at 1:200 (wt/wt)(FIG. 3C), 1:500 (wt/wt) (FIG. 3D), and 1:750 (wt/wt) (FIG. 3E).

FIG. 4 is a representation of a gel electrophoresis of native(unmodified) siRNA (FIG. 4A) and siRNA-S-S-PE/PEG-PE mixed micelles(1:750 wt/wt) (FIG. 4B) either before or after incubation with RNAse.

FIG. 5 is a series of schematic representations of scans showing GFPsilencing in C166-GFP endothelial cells by control micelles (FIG. 5A),naked siRNA (FIG. 5B), and siRNA-S-S-PE/PEG-PE mixed micellepreparations (1:750 wt/wt) (FIG. 5C) measured by flow cytometry.

FIG. 6A is a graphic representation of GFP-silencing in GFP-C166endothelial cells by siRNA-S-S-PE/PEG-PE mixed micelles (1:750) andsiRNA/Lipofectamine as measured by flow cytometry.

FIG. 6B is a graphic representation of cell viability after a 48 hincubation with various siRNA formulations.

DESCRIPTION

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting. Unless otherwisedefined, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs. Although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention, suitable methods and materials aredescribed below.

DEFINITIONS

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

The term “about” is used herein to mean a value − or +20% of a givennumerical value. Thus, “about 60%” means a value of between 60−(20% of60) and 60+(20% of 60) (i.e., between 48 and 70).

The terms “peptide”, “polypeptide” and “protein” are usedinterchangeably herein.

The term “pharmaceutically effective amount” or “therapeuticallyeffective amount” refers to an amount (e.g., dose) effective in treatinga patient, having a disorder or condition described herein. It is alsoto be understood herein that a “pharmaceutically effective amount” maybe interpreted as an amount giving a desired therapeutic effect, eithertaken in one dose or in any dosage or route, taken alone or incombination with other therapeutic agents.

The term “treatment” or “treating”, as used herein, refers toadministering a therapy in an amount, manner, and/or mode effective toimprove a condition, symptom, or parameter associated with a disorder orcondition or to prevent or reduce progression of a disorder orcondition, either to a statistically significant degree or to a degreedetectable to one skilled in the art. An effective amount, manner, ormode can vary depending on the subject and may be tailored to thesubject.

The term “subject”, as used herein, means any subject for whomdiagnosis, prognosis, or therapy is desired. For example, a subject canbe a mammal, e.g., a human or non-human primate (such as an ape, monkey,orangutan, or chimpanzee), a dog, cat, guinea pig, rabbit, rat, mouse,horse, cattle, or cow.

As used herein, the term “antibody” refers to a polypeptide thatincludes at least one immunoglobulin variable region, e.g., an aminoacid sequence that provides an immunoglobulin variable domain orimmunoglobulin variable domain sequence. For example, an antibody caninclude a heavy (H) chain variable region (abbreviated herein as VH),and a light (L) chain variable region (abbreviated herein as VL). Inanother example, an antibody includes two heavy (H) chain variableregions and two light (L) chain variable regions. The term “antibody”encompasses antigen-binding fragments of antibodies (e.g., single chainantibodies, Fab, F(ab′)₂, Fd, Fv, and dAb fragments) as well as completeantibodies, e.g., intact immunoglobulins of types IgA, IgG, IgE, IgD,IgM (as well as subtypes thereof). The light chains of theimmunoglobulin can be of types kappa or lambda. In one embodiment, theantibody is glycosylated.

As used herein, the terms “coupled”, “linked”, “fused”, and “fusion” areused interchangeably. These terms refer to the joining together of twomore elements or components by whatever means, including chemicalconjugation or recombinant means.

An “RNA interfering agent”, as used herein, is defined as any agent thatinterferes with or inhibits expression of a target gene or genomicsequence by RNA interference (RNAi). Such RNA interfering agentsinclude, but are not limited to, nucleic acid molecules including RNAmolecules that are homologous to the target gene or genomic sequence, ora fragment thereof, short interfering RNA (siRNA), and small moleculesthat interfere with or inhibit expression of a target gene by RNAinterference (RNAi).

“RNA interference” or “RNAi”, as used herein, is an evolutionallyconserved process whereby the expression or introduction of RNA of asequence that is identical or highly similar to a target gene results inthe sequence specific degradation or specific post-transcriptional genesilencing (PTGS) of messenger RNA (mRNA) transcribed from that targetedgene (see Coburn, G. and Cullen, B. (2002) J. of Virology 76(18):9225),thereby inhibiting expression of the target gene. In one embodiment, theRNA is double stranded RNA (dsRNA). This process has been described inplants, invertebrates, and mammalian cells. In nature, RNAi is initiatedby the dsRNA-specific endonuclease Dicer, which promotes processivecleavage of long dsRNA into double-stranded fragments termed siRNAs.siRNAs are incorporated into a protein complex that recognizes andcleaves target mRNAs. RNAi can also be initiated by introducing nucleicacid molecules, e.g., synthetic siRNAs or RNA interfering agents, toinhibit or silence the expression of target genes.

“Short interfering RNA”, “small interfering RNA” or “siRNA”, as usedherein, is an agent that functions to inhibit expression of a targetgene, e.g., by RNAi. An siRNA can be chemically synthesized, can beproduced by in vitro transcription, or can be produced within a hostcell. In one embodiment, siRNA is a double stranded RNA (dsRNA) moleculeof about 15 to about 40 nucleotides in length, of about 15 to about 28nucleotides, of about 19 to about 25 nucleotides in length, or about 19,20, 21, or 22 nucleotides in length, and can contain a 3′ and/or 5′overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5nucleotides. The length of the overhang is independent between the twostrands, i.e., the length of the over hang on one strand is notdependent on the length of the overhang on the second strand. The siRNAis capable of promoting RNA interference, e.g., through degradation orspecific post-transcriptional gene silencing (PTGS) of the targetmessenger RNA (mRNA).

As used herein, “inhibition of target gene expression” includes anydecrease in expression or protein activity or level of the target geneor protein encoded by the target gene. The decrease can be of at leastabout 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more, as comparedto the expression of a target gene or the activity or level of theprotein encoded by a target gene that has not been targeted by an RNAinterfering agent.

General

The disclosure is based, in part, on the discovery that an siRNAreversibly conjugated to a phospholipid of a micelle can be releasedfrom the phospholipid when exposed to reducing conditions, and thereleased siRNA can inhibit expression of a target polypeptide. While notwishing to be bound by theory, it is believed that the conjugated siRNAcompositions described herein have increased stability relative to thesiRNA when not reversibly conjugated to a phospholipid. For example,siRNA, when incorporated into a conjugated siRNA composition describedherein, can be protected from degradation (e.g., enzymes such as RNase)and can have higher circulation times when administered into a subject.Liposomes and micelles containing the reversibly conjugated siRNA areuseful for the delivery of siRNA to target cells and tissues to reduceor inhibit the expression of a target polypeptide, e.g., to treat adisease or a disorder.

siRNA

The present disclosure relates to siRNA molecules of about 15 to about40, about 20 to about 40, or about 15 to about 28 nucleotides in length,which are homologous to a target gene or sequence and mediate RNAi ofthe target gene or sequence. The siRNA molecules can have a length ofabout 19 to about 25 nucleotides, such as a length of about 19, 20, 21,or 22 nucleotides. The siRNA molecules can also comprise a 3′ hydroxylgroup. The siRNA molecules can be single-stranded or double stranded;such molecules can be blunt ended or comprise overhanging ends (e.g.,5′, 3′). In specific embodiments, the RNA molecule is double strandedand either blunt ended or comprises overhanging ends.

In some instances, at least one strand of the RNA molecule has a 3′overhang from about 0 to about 6 nucleotides (e.g., pyrimidinenucleotides, purine nucleotides) in length. In other situations, the 3′overhang is from about 1 to about 5 nucleotides, from about 1 to about 3nucleotides, from about 2 to about 4 nucleotides, from about 3 to about6 nucleotides, or from about 2 to about 5 nucleotides in length. In oneinstance, the RNA molecule is double-stranded, one strand has a 3′overhang and the other strand can be blunt-ended or have an overhang.When the RNA molecule is double stranded and both strands comprise anoverhang, the length of the overhangs can be the same or different foreach strand. In a particular instance, the RNA comprises about 19, 20,21, or 22 nucleotide strands that are paired and that have overhangs offrom about 1 to about 3, particularly about 2, nucleotides on both 3′ends of the RNA. The 3′ overhangs can be stabilized against degradation.In some instances, the RNA is stabilized by including purinenucleotides, such as adenosine or guanosine nucleotides. Alternatively,substitution of pyrimidine nucleotides by modified analogues, e.g.,substitution of uridine 2 nucleotide 3′ overhangs, by 2′-deoxythymidinecan be used.

siRNA molecules useful in the methods described herein are not limitedto those molecules containing only RNA, but can also encompasschemically modified nucleotides and non-nucleotides, and also includemolecules wherein a ribose sugar molecule is substituted for anothersugar molecule or a molecule which performs a similar function.Moreover, a non-natural linkage between nucleotide residues may be used,such as a phosphorothioate, phosphorodithioate, alkylphosphonate,phosphoramidate, carbamate, phosphatetriester, alkylphosphonothioate,and/or acetamidate linkage. The RNA strand can be derivatized with areactive functional group of a reporter group, such as a fluorophore.Particularly useful derivatives are modified at a terminus or termini ofan RNA strand, such as the 3′ terminus of the sense strand. For example,the 2′-hydroxyl at the 3′ terminus can be readily and selectivelyderivatized with a variety of groups.

Other useful RNA derivatives incorporate nucleotides having modifiedcarbohydrate moieties, such as 2′-O-substituted moieties (such as2′O-alkylated residues or 2′-O-methyl ribosyl derivatives and2′-O-fluoro ribosyl derivatives).

The RNA bases may also be modified. Any modified base useful forinhibiting or interfering with the expression of a target sequence maybe used. For example, halogenated bases, such as 5-bromouracil and5-iodouracil can be incorporated. The bases may also be alkylated, forexample, 7-methylguanosine can be incorporated in place of a guanosineresidue.

The target gene can be a gene or sequence of a cellular gene, viralgene, or infectious gene, or a fragment thereof. An siRNA can besubstantially homologous to the target gene or genomic sequence, or afragment thereof. As used herein, the term “homologous” is defined asbeing substantially identical, sufficiently complementary, or similar tothe target mRNA, or a fragment thereof, to effect RNA interference ofthe target. Non-natural bases that yield successful inhibition can alsobe incorporated.

Synthetic siRNA molecules can be obtained using a number of techniquesknown to those of skill in the art. For example, the siRNA molecule canbe chemically synthesized or recombinantly produced using methods knownin the art, such as using appropriately protected ribonucleosidephosphoramidites and a conventional DNA/RNA synthesizer (see, e.g.,Elbashir et al., Nature 411:494-498 (2001); Elbashir et al., Genes &Development 15:188-200 (2001); Harborth et al., J. Cell Science114:4557-4565 (2001); Masters et al., Proc. Natl. Acad. Sci. (USA)98:8012-8017 (2001); and Tuschl et al., Genes & Development 13:3191-3197(1999)). Alternatively, several commercial RNA synthesis suppliers areavailable including, but not limited to, Proligo (Hamburg, Germany),Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part ofPerbio Science, Rockford, Ill., USA), Glen Research (Sterling, Va.,USA), ChemGenes (Ashland, Mass., USA), and Cruachem (Glasgow, UK). Assuch, siRNA molecules are not difficult to synthesize and are readilyprovided in a quality suitable for RNAi.

The targeted region of an siRNA molecule can be selected from a giventarget gene sequence, e.g., a cellular or viral target sequence,beginning from about 25 to 50 nucleotides, from about 50 to 75nucleotides, or from about 75 to 100 nucleotides downstream of the startcodon. Nucleotide sequences can contain 5′ or 3′ UTRs and regions nearbythe start codon. One method of designing a siRNA molecule involvesidentifying the 23 nucleotide sequence motif AA(N19)TT (where N can beany nucleotide) and selecting sequences with at least 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% G/C content. Alternatively, ifno such sequence is found, the search can be extended using the motifNA(N21), where N can be any nucleotide. In this situation, the 3′ end ofthe sense siRNA can be converted to TT to allow for the generation of asymmetric duplex with respect to the sequence composition of the senseand antisense 3′ overhangs. The antisense siRNA molecule may then besynthesized as the complement to nucleotide positions 1 to 21 of the 23nucleotide sequence motif. The use of symmetric 3′ TT overhangs can beused so that the small interfering ribonucleoprotein particles (siRNPs)are formed with approximately equal ratios of sense and antisense targetRNA-cleaving siRNPs (Elbashir et al. (2001) supra and Elbashir et al.2001 supra). Analysis of sequence databases, including but not limitedto the NCBI, BLAST, Derwent and GenSeq, as well as commerciallyavailable oligosynthesis companies such as Oligoengine™, can also beused to select siRNA sequences against EST libraries to ensure that onlyone gene is targeted.

Liposomes

The present disclosure includes siRNA molecules reversibly conjugated toa phospholipid of a liposome. Upon exposure to reducing conditions, suchas within a cell, the siRNA is unconjugated from the phospholipid of theliposome and can inhibit expression of a target polypeptide. Liposomesare vesicles that include one or more concentrically ordered lipidbilayer(s) encapsulating an aqueous phase, when in an aqueousenvironment. Such vesicles are formed in the presence of“vesicle-forming lipids”, which are defined herein as amphipathic lipidscapable of either forming or being incorporated into a bilayerstructure. The term includes lipids that are capable of forming abilayer by themselves or when in combination with another lipid orlipids. An amphipathic lipid is incorporated into a lipid bilayer byhaving its hydrophobic moiety in contact with the interior, hydrophobicregion of the bilayer membrane and its polar head moiety orientedtowards an outer, polar surface of the membrane. Hydrophilicity arisesfrom the presence of functional groups, such as hydroxyl, phosphate,carboxyl, sulfate, amino or sulfhydryl groups. Hydrophobicity resultsfrom the presence of a long chain of aliphatic hydrocarbon groups.

Liposomes include multilamellar vesicles, multivesicular liposomes,unilamellar vesicles, and giant liposomes. Multilamellar liposomes (alsoknown as multilamellar vesicles (“MLV”)) contain multiple concentricbilayers within each liposome particle, resembling the layers of anonion. Multivesicular liposomes consist of lipid membranes enclosingmultiple non-concentric aqueous chambers. Unilamellar liposomes enclosea single internal aqueous compartment. Single bilayer (or substantiallysingle bilayer) liposomes include small unilamellar vesicles (“SUV”) andlarge unilamellar vesicles (“LUV”). LUVs and SUVs can range in size fromabout 50 nm to about 500 nm and about 20 nm to about 50 nm,respectively. Giant liposomes can range in size from about 5000 nm toabout 50,000 nm (Needham et al., Colloids and Surfaces B: Biointerfaces18:183-195 (2000)).

Any suitable vesicle-forming lipid (e.g., naturally occurring lipids andsynthetic lipids) can be utilized in the liposomes and micellesdescribed herein. Suitable lipids include, without limitation,phospholipids such as phosphatidylcholine (PC), phosphatidylglycerol(PG), phosphatidylinositol (PI), phosphatidic acid (PA),phosphatidyethanolamine (PE), phosphatidylserine (PS), andphosphoethanolamine; sterols such as cholesterol; glycolipids;sphingolipids such as sphingosine, ceramides, sphingomyelin, andglycosphingolipids (such as cerebrosides and gangliosides). Particularlipids include dipalmitoyl phosphatidylcholine, cholesterol,ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine,sodium cholate, dicetyl phosphatidylethanolamine-polyglycerin 8G,dimyristoyl phosphatidylcholine, distearoyl phosphatidylcholine,dioleoyl phosphatidylcholine, dimyristoyl phosphatidylserine,dipalmitoyl phosphatidylserine, distearoyl phosphatidylserine, dioleoylphosphatidylserine, dimyristoyl phosphatidylinositol, dipalmitoylphosphatidylinositol, distearoyl phosphatidylinositol, dioleoylphosphatidylinositol, dimyristoyl phosphatidylethanolamine, distearoylphosphatidylethanolamine, distearoyl phosphoethanolamine, dioleoylphosphatidylethanolamine, dimyristoyl phosphatidic acid, dipalmitoylphosphatidic acid, distearoyl phosphatidic acid, dioleoyl phosphatidicacid, galactosyl ceramides, glycosyl ceramides, lactosyl ceramides,phosphatides, globosides, GM1 (Galβ1, 3GalNAcβ1, 4(NeuAa2,3)Galβ1,4Glcβ1, l'Cer), ganglioside GD1a, ganglioside GD1b, dimyristoylphosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoylphosphatidylglycerol, dioleoyl phosphatidylglycerol,distearoyl-glycero-phosphoethanolamine, and1,2-dioleoyl-sn-glycero-3-phosphoethanolamine. Suitable phospholipidscan include one or two acyl chains having any number of carbon atoms,such as about 6 to about 24 carbon atoms, selected independently of oneanother and with varying degrees of unsaturation. Thus, combinations ofphospholipid of different species and different chain lengths in varyingratios can be used. Mixtures of lipids in suitable ratios, as judged byone of skill in the art, can also be used.

Particular phospholipids useful in the methods described herein are1,2-didecanoyl-sn-glycero-3-phosphoethanolamine,1,2-dilauroyl-sn-glycero-3-phosphoethanolamine,1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine,1,2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine,1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, and1,2-distearoyl-sn-glycero-3-phosphoethanolamine.

Liposomes can be generated using a variety of techniques known in theart. These techniques include, without limitation, ether injection(Deamer et al., Acad. Sci. 308:250 (1978)); surfactant (Brunner et al.,Biochim. Biophys. Acta 455:322 (1976)); Ca²⁺ fusion (Paphadjopoulos etal., Biochim. Biophys. Acta 394:483 (1975)); freeze-thaw (Pick et al.,Arach. Biochim. Biophys. 212:186 (1981)); reverse-phase evaporation(Szoka et al., Biochim. Biophys. Acta 601:559 (1980)); ultrasonictreatment (Huang et al., Biochem. 8:344 (1969)); ethanol injection(Kremer et al., Biochem. 16:3932 (1977)); extrusion (Hope et al.,Biochim. Biophys. Acta 812:55 (1985)); French press (Barenholz et al.,FEBS Lett. 99:210 (1979)); thin film hydration (Bangham et al., J. Mol.Biol. 13:238-252 (1965)); and any other methods described herein orknown in the art. Liposomes can also be generated using commerciallyavailable kits (e.g., from Boehringer-Mannheim, ProMega, and LifeTechnologies (Gibco)).

Different techniques can be used depending on the type of liposomedesired. For example, small unilamellar vesicles (SUVs) can be preparedby the ultrasonic treatment method, the ethanol injection method, or theFrench press method, while multilamellar vesicles (MLVs) can be preparedby the reverse-phase evaporation method or by the simple addition ofwater to a lipid film, followed by dispersal by mechanical agitation(Bangham et al., J. Mol. Biol. 13:238-252 (1965)). LUVs can be preparedby the ether injection method, the surfactant method, the Ca²⁺ fusionmethod, the freeze-thaw method, the reverse-phase evaporation method,the French press method, or the extrusion method.

Average liposome size can be determined by known techniques, such asquasi-elastic light scattering, photon correlation spectroscopy, dynamiclight scattering, or various electron microscopy techniques (such asnegative staining transmission electron microscopy, freeze fractureelectron microscopy or cryo-transmission electron microscopy). In someinstances, the resulting liposomes can be run down a Sephadex™ G50column or similar size exclusion chromatography column equilibrated withan appropriate buffer in order to remove unencapsulated therapeuticagents or detection agents described herein.

Liposomes can range in size, such as from about 50 nm to about 1 μm indiameter. For example, liposomes described herein can be less than about200 nm in diameter, less than about 160 nm in diameter, or less thanabout 140 nm in diameter. In some embodiments, liposomes describedherein can be substantially uniform in size, for example, 10% to 100%,or more generally at least 10%, 20%, 30%, 40%, 50, 55% or 60%, or atleast 65%, 75%, 80%, 85%, 90%, or 95%, or as much as 96%, 97%, 98%, 99%,or 100% of the liposomes can have the same size. In some instances,liposomes can be sized by extrusion through a filter (e.g., apolycarbonate filter) having pores or passages of the desired diameter.

In some instances, liposomes can include a hydrophilic moiety. Attachinga hydrophilic moiety to the surface of liposomes can stericallystabilize liposomes and can increase the circulation longevity of theliposome. This can enhance blood stability and increase circulationtime, reduce uptake into healthy tissues, and increase delivery todisease sites such as solid tumors (see, e.g., U.S. Pat. Nos. 5,013,556and 5,593,622; and Patel et al., Crit. Rev. Ther. Drug Carrier Syst.9:39 (1992)). The hydrophilic moiety can be conjugated to a lipidcomponent of the liposome, forming a hydrophilic polymer-lipidconjugate. The term “hydrophilic polymer-lipid conjugate”, as usedherein, refers to a lipid (e.g., a vesicle-forming lipid) covalentlyjoined at its polar head moiety to a hydrophilic polymer, and can bemade by attaching the polymer to a reactive functional group at thepolar head moiety of the lipid. The covalent linkage can be releasable,such that the polymer dissociates from the lipid (at, e.g.,physiological pH or after a variable length of time (see, e.g.,Adlakha-Hutcheon et al., Nat. Biotechnol. 17:775-779 (1999)).Nonlimiting suitable reactive functional groups include, e.g., amino,hydroxyl, carboxyl, and formyl groups. The lipid can be any lipiddescribed in the art for use in such conjugates. For example, the lipidcan be a phospholipid having one or two acyl chains including betweenabout 6 to about 24 carbon atoms in length with varying degrees ofunsaturation.

In some circumstances, the lipid in the conjugate can be aphosphatidyethanolamine, such as of the distearoyl form. The polymer canbe a biocompatible polymer. In some instances, the polymer has asolubility in water that permits polymer chains to extend away from aliposome surface with sufficient flexibility that produces uniformsurface coverage of a liposome. Such a polymer can be a polyalkylether,including PEG, polymethylene glycol, polyhydroxy propylene glycol,polypropylene glycol, polylactic acid, polyglycolic acid, polyacrylicacid and copolymers thereof, as well as those disclosed in U.S. Pat.Nos. 5,013,556 and 5,395,619. The polymer can have an average molecularweight between about 350 daltons and about 10,000 daltons.

Micelles

The present disclosure includes siRNA molecules reversibly conjugated toa phospholipid of a micelle. One exemplary micelle is depicted in FIG.2A. Upon exposure to reducing conditions, such as within a cell, thesiRNA is unconjugated from the phospholipid of the micelle and caninhibit expression of a target polypeptide. Micelles are vesicles thatinclude a single lipid monolayer encapsulating an aqueous phase.Micelles can be spherical or tubular and form spontaneously about thecritical micelle concentration (“CMC”). In general, micelles are inequilibrium with the monomers under a given set of physical conditionssuch as temperature, ionic environment, concentration, etc.

Micelles are formed in the presence of “micelle-forming compounds”,which include amphipathic lipids (e.g., a vesicle-forming lipid asdescribed herein or known in the art), lipoproteins, detergents,non-lipid polymers, or any other compound capable of either forming orbeing incorporated into a monolayer vesicle structure. Thus, amicelle-forming compound includes compounds that are capable of forminga monolayer by themselves or when in combination with another compound,and may be polymer micelles, block co-polymer micelles, polymer-lipidmixed micelles, or lipid micelles. A micelle-forming compound, in anaqueous environment, generally has a hydrophobic moiety in contact withthe interior of the vesicle, and a polar head moiety oriented outwardsinto the aqueous environment. Hydrophilicity generally arises from thepresence of functional groups, such as hydroxyl, phosphate, carboxyl,sulfate, amino or sulfhydryl groups. Hydrophobicity generally resultsfrom the presence of a long chain of aliphatic hydrocarbon groups.

A micelle can be prepared, e.g., from lipoproteins or artificiallipoproteins including low density lipoproteins, chylomicrons and highdensity lipoproteins. Micelles can be generated using a variety of knowntechniques, including, without limitation, simple dispersion by mixingin aqueous or hydroalcoholic media or media containing surfactants orionic substances; sonication; solvent dispersion; or any other techniquedescribed herein or known in the art. Different techniques can be used,depending on the type of micelle desired and the physicochemicalproperties of the micelle-forming components, such as solubility,hydrophobicity and behavior in ionic or surfactant-containing solutions.

Micelles can range in size, such as between about 5 nm to about 50 nm indiameter. In some instances, micelles can be less than about 50 nm indiameter, less than about 30 nm in diameter, or less than about 20 nm indiameter.

In some situations, micelles described herein can include a hydrophilicpolymer-lipid conjugate, as described herein or known in the art.

Conjugation

The siRNA can be specifically modified such that it can be conjugated tothat phospholipid. For example, one or more nucleotides of an siRNA canbe modified to include a carboxylic acid functionality, and aphospholipid can be modified with an amine. The amine would then becapable of reacting with the carboxylic acid functionality on the siRNA,using procedures known in the art, to form an amide linkage between thephospholipid and siRNA. Alternatively, the siRNA can be modified with anamine and reacted with a carboxylic functionality on a phospholipid toform an amide linkage. Nonlimiting examples of other functionalitiesthat react with amines are acyl chlorides, acid anhydrides, esters, andcarboxylic salts.

The siRNA-phospholipid conjugate can be linked, for example, via amidelinkages. One particular mechanism of producing carbamide linkagesincludes the use of alkyl chloroformate groups. In one such nonlimitingexample, a phospholipid with a chloroformate group is reacted with ansiRNA having an amine group functionality in the presence of a base. Theresulting compound is a siRNA-phospholipid conjugated by a carbamidelinkage.

An siRNA can also be conjugated to a phospholipid via an ester bond. Acommon method of performing such esterifications is the use of Steglichesterification. In this example, siRNAs are modified with carboxylicacid functionalities at one or more positions. The carboxylic acids arethen activated with dicyclohexylcarbodiimide. Subsequently,4-dimethylaminopyridine is used to catalyze an acyl-transfer with ahydroxyl group on the phospholipid.

In addition, siRNAs can be conjugated to a phospholipid via an etherlinkage. Such a linkage can be made using procedures known in the art,such as Williamson ether synthesis reaction. The Williamson reactioninvolves using sodium hydroxide or another base to form an alkoxide ofan alcohol on the siRNA. The phospholipid to be linked can be analiphatic compound bearing a suitable leaving group, such as an iodide,bromide, or sulfonate leaving group.

Another useful conjugate is an siRNA disulfide linked to a phospholipid,such as an siRNA-S-S-PE (e.g.,1,2-Dipalmitoyl-sn-Glycero-3-Phosphothioethanol). By activating the3′-sense strand of a hexylamine-modified-siRNA with the pyridyldisulfidecontaining crosslinker N-succinimidyl-3-(2-pyridyldithio)propionate(SPDP), the activated siRNA-PDP reacts with the —SH group of thephosphothiethanol (PE-SH) forming a reversible disulfide bond (accordingto FIG. 2). Another useful pyridyl disulfide-containing crosslinker is 4saccinimidyloxycarbonyl-x-methyl-x-(2-pyridyldithio) toluene (SMPT).

One distinguishing characteristic of this conjugate is the chemicalbinding between the siRNA and a phospholipid (PE-SH), instead ofclassical binding based on electrostatic interactions between negativeand positive charges respectively. The chemical conjugation improves thesiRNA stability against nucleases attack, but also provides a reversiblebond able to be hydrolyzed into the natural cytosolic environment,freeing the siRNA to act into the cells. The technique can be applied toany kind of siRNA which mediates the knock-down of different genes.

Detection Agents

In some instances, the liposomes or micelles described herein, e.g., aliposome or micelle reversibly conjugated to an siRNA, can be used todetect or image cells, e.g., using a liposome or micelle that includes adetection agent. The detection agent can be used to qualitatively orquantitatively analyze the location and/or the amount of a liposome ormicelle at a particular locus. The detection agent can also be used toimage a liposome, micelle, and/or a cell or tissue target of a liposomeor micelle using standard methods.

A liposome or micelle described herein can be derivatized (or labeled)with a detection agent by attaching the agent to a component or aphospholipid of the liposome or micelle. Nonlimiting examples ofdetection agents include, without limitation, fluorescent compounds,various enzymes, prosthetic groups, luminescent materials,bioluminescent materials, fluorescent emitting metal atoms, (e.g.,europium (Eu)), radioactive isotopes (described below), quantum dots,electron-dense reagents, and haptens. The detection reagent can bedetected using various means including, but are not limited to,spectroscopic, photochemical, radiochemical, biochemical,immunochemical, or chemical means.

Nonlimiting exemplary fluorescent detection agents include fluorescein,fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and thelike. A detection agent can also be a detectable enzyme, such asalkaline phosphatase, horseradish peroxidase, β-galactosidase,acetylcholinesterase, glucose oxidase and the like. When a liposome ormicelle is derivatized with a detectable enzyme, it can be detected byadding additional reagents that the enzyme uses to produce a detectablereaction product. For example, when the detection agent is horseradishperoxidase, the addition of hydrogen peroxide and diaminobenzidine leadsto a detectable colored reaction product. A liposome or micelle can alsobe derivatized with a prosthetic group (e.g., streptavidin/biotin andavidin/biotin). For example, a liposome or micelle can be derivatizedwith biotin and detected through indirect measurement of avidin orstreptavidin binding. Nonlimiting examples of fluorescent compounds thatcan be used as detection reagents include umbelliferone, fluorescein,fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride, and phycoerythrin. Luminescent materialsinclude, e.g., luminol, and bioluminescent materials include, e.g.,luciferase, luciferin, and aequorin.

A detection agent can also be a radioactive isotope, such as, but notlimited to, α-, β-, or γ-emitters; or β- and γ-emitters. Radioactiveisotopes can be used in diagnostic or therapeutic applications. Suchradioactive isotopes include, but are not limited to, iodine (¹³¹I or¹²⁵I), yttrium (⁹⁰Y), lutetium (¹⁷⁷Lu), actinium (²²⁵Ac), praseodymium(¹⁴²PR or ¹⁴³Pr), astatine (²¹¹At), rhenium (¹⁸⁶Re or ¹⁸⁷Re), bismuth(²¹²Bi or ²¹³Bi), indium (¹¹¹In), technetium (^(99m)Tc), phosphorus(³²P), rhodium (¹⁸⁸Rh), sulfur (³⁵S), carbon (¹⁴C), tritium (³H),chromium (⁵¹Cr), chlorine (³⁶Cl), cobalt (⁵⁷Co or ⁵⁸Co), iron (⁵⁹Fe),selenium (⁷⁵Se), and gallium (⁶⁷Ga).

The liposomes or micelles can be radiolabeled using techniques known inthe art. In some situations, a liposome or micelle described herein iscontacted with a chelating agent, e.g.,1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA), tothereby produce a conjugated liposome or micelle. The conjugatedliposome or micelle is then radiolabeled with a radioisotope, e.g.,¹¹¹In, ⁹⁰Y, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁷Re, or ^(99m)Tc, to thereby produce alabeled liposome or micelle. In other methods, the liposome or micellecan be labeled with ¹¹¹In and ⁹⁰Y using weak transchelators such ascitrate (see, e.g., Khaw et al., Science 209:295-297 (1980)) or ^(99m)Tcafter reduction in reducing agents such as Na Dithionite (see, e.g.,Khaw et al., J. Nucl. Med. 23:1011-1019 (1982)) or by SnCl₂ reduction(see, e.g., Khaw et al., J. Nucl. Med. 47:868-876 (2006)). Other methodsare described in, e.g., Lindegren et al., Bioconjug. Chem. 13:502-509(2002); Boyd et al., Mol. Pharm. 3:614-627 (2006); and del Rosario etal., J. Nucl. Med. 34:1147-1151 (1993).

Targeting Agents

The conjugated siRNA composition can include a targeting agent, e.g.,attached to a phospholipid of the liposome or micelle of thecomposition, or to an siRNA and/or PEG of the composition. The targetingagents can be, for example, various specific ligands, such asantibodies, monoclonal antibodies and their fragments, folate, mannose,galactose and other mono-, di-, and oligosaccharides, and RGD peptide.

The liposomes and micelles of the compositions described herein are notlimited to any particular targeting agent, and a variety of targetingagents can be used. Examples of such targeting agents include, but arenot limited to, nucleic acids (e.g., RNA and DNA), polypeptides (e.g.,receptor ligands, signal peptides, avidin, Protein A, and antigenbinding proteins), polysaccharides, biotin, hydrophobic groups,hydrophilic groups, drugs, and any organic molecules that bind toreceptors. In some instances, a liposome or micelle described herein canbe conjugated to one, two, or more of a variety of targeting agents. Forexample, when two or more targeting agents are used, the targetingagents can be similar or dissimilar. Utilization of more than onetargeting agent on a particular liposome or micelle can allow thetargeting of multiple biological targets or can increase the affinityfor a particular target.

The targeting agents can be associated with the liposomes or micellesreversibly conjugated to an siRNA in a number of ways. For example, thetargeting agents can be associated (e.g., covalently or noncovalentlybound) to a phospholipid of the liposome or micelle with either short(e.g., direct coupling), medium (e.g., using small-molecule bifunctionallinkers such as SPDP (Pierce Biotechnology, Inc., Rockford, Ill.)), orlong (e.g., PEG bifunctional linkers (Nektar Therapeutics, Inc., SanCarlos, Calif.)) linkages.

In addition, a liposome or micelle reversibly conjugated to an siRNA canalso incorporate reactive groups (e.g., amine groups such as polylysine,dextranemine, profamine sulfate, and/or chitosan). The reactive groupcan allow for further attachment of various specific ligands or reportergroups (e.g., ¹²⁵I, ¹³¹I, I, Br, various chelating groups such as DTPA,which can be loaded with reporter heavy metals such as ¹¹¹In, ^(99m)Tc,Gd, Mn, fluorescent groups such as FITC, rhodamine, Alexa, and quantumdots), and/or other moieties (e.g., ligands, antibodies, and/or portionsthereof).

Antibodies as Targeting Agents

In some instances, the targeting agents for a liposome or micellereversibly conjugated to an siRNA are antigen binding proteins orantibodies or binding portions thereof. Antibodies can be generated toallow for the specific targeting of antigens or immunogens (e.g., tumor,tissue, or pathogen specific antigens) on various biological targets(e.g., pathogens, tumor cells, normal tissue). Such antibodies include,but are not limited to, polyclonal antibodies; monoclonal antibodies orantigen binding fragments thereof; modified antibodies such as chimericantibodies, reshaped antibodies, humanized antibodies, or fragmentsthereof (e.g., Fv, Fab′, Fab, F(ab′)₂); or biosynthetic antibodies,e.g., single chain antibodies, single domain antibodies (DAB), Fvs, orsingle chain Fvs (scFv).

In certain instances, the targeting agent is an antibody thespecifically binds an angiogenesis agent described herein. For example,the targeting agent is an anti-VEGF antibody described herein, e.g.,bevacizumab. In other examples, the liposome or micelle includes, inaddition to an anti-angiogenesis agent described herein, an additionalantibody that targets additional ligands.

Methods of making and using polyclonal and monoclonal antibodies arewell known in the art, e.g., in Harlow et al., Using Antibodies: ALaboratory Manual: Portable Protocol I. Cold Spring Harbor Laboratory(Dec. 1, 1998). Methods for making modified antibodies and antibodyfragments (e.g., chimeric antibodies, reshaped antibodies, humanizedantibodies, or fragments thereof, e.g., Fab′, Fab, F(ab′)₂ fragments);or biosynthetic antibodies (e.g., single chain antibodies, single domainantibodies (DABs), Fv, single chain Fv (scFv), and the like), are knownin the art and can be found, e.g., in Zola, Monoclonal Antibodies:Preparation and Use of Monoclonal Antibodies and Engineered AntibodyDerivatives, Springer Verlag (Dec. 15, 2000; 1st edition).

Antibody attachment can be performed via any method that does notcompromise the ability of the antibody to target a tumor, and to treatit, e.g., by binding to a specific anti-angiogenesis factor. Forexample, attachment can be performed through standard covalent bindingto free amine groups (see, e.g., Torchilin et al., Hybridoma 6:229-240(1987); Torchilin et al, Biochim. Biophys. Acta 1511:397-411 (2001);Masuko et al., Biomacromol. 6:800-884 (2005)).

Signal Peptides as Targeting Agents

In some instances, a targeting agent for a liposome or micellereversibly conjugated to an siRNA can be a signal peptide. Thesepeptides can be chemically synthesized or cloned, expressed and purifiedusing known techniques. Signal peptides can be used to target theliposomes or micelles described herein to a target cell or tissue.

Nucleic Acids as Targeting Agents

In other instances, the targeting agent for a liposome or micellereversibly conjugated to an siRNA is a nucleic acid (e.g., RNA or DNA).In some examples, the nucleic acid targeting agents are designed tohybridize by base pairing to a particular nucleic acid (e.g.,chromosomal DNA, mRNA, or ribosomal RNA). In other situations, thenucleic acids bind a ligand or biological target. For example, thenucleic acid can bind reverse transcriptase, Rev or Tat proteins of HIV(Tuerk et al., Gene 137:33-9 (1993)); human nerve growth factor (Binkleyet al., Nuc. Acids Res. 23:3198-205 (1995)); or vascular endothelialgrowth factor (Jellinek et al., Biochem. 83:10450-10456 (1994)). Nucleicacids that bind ligands can be identified by known methods, such as theSELEX procedure (see, e.g., U.S. Pat. Nos. 5,475,096; 5,270,163; and5,475,096; and WO 97/38134; WO 98/33941; and WO 99/07724). The targetingagents can also be aptamers that bind to particular sequences.

Other Targeting Agents

The targeting agents for a liposome or micelle reversibly conjugated toan siRNA can recognize a variety of epitopes on preselected biologicaltargets (e.g., pathogens, tumor cells, or normal cells). For example, insome instances, the targeting agent can be sialic acid to target HIV(Wies et al., Nature 333:426 (1988)), influenza (White et al., Cell56:725 (1989)), Chlamydia (Infect. Immunol. 57:2378 (1989)), Neisseriameningitidis, Streptococcus suis, Salmonella, mumps, newcastle,reovirus, Sendai virus, and myxovirus; and 9-OAC sialic acid to targetcoronavirus, encephalomyelitis virus, and rotavirus; non-sialic acidglycoproteins to target cytomegalovirus (Virol. 176:337 (1990)) andmeasles virus (Virol. 172:386 (1989)); CD4 (Khatzman et al., Nature312:763 (1985)), vasoactive intestinal peptide (Sacerdote et al., J.Neurosci. Res. 18:102 (1987)), and peptide T (Ruff et al., FEBS Letters211:17 (1987)) to target HIV; epidermal growth factor to target vaccinia(Epstein et al., Nature 318:663 (1985)); acetylcholine receptor totarget rabies (Lentz et al., Science 215:182 (1982)); Cd3 complementreceptor to target Epstein-Barr virus (Carel et al., J. Biol. Chem.265:12293 (1990)); beta-adrenergic receptor to target reovirus (Co etal., Proc. Natl. Acad. Sci. USA 82:1494 (1985)); ICAM-1 (Marlin et al.,Nature 344:70 (1990)), N-CAM, and myelin-associated glycoprotein MAb(Shephey et al., Proc. Natl. Acad. Sci. USA 85:7743 (1988)) to targetrhinovirus; polio virus receptor to target polio virus (Mendelsohn etal., Cell 56:855 (1989)); fibroblast growth factor receptor to targetherpes virus (Kaner et al., Science 248:1410 (1990)); oligomannose totarget Escherichia coli; and ganglioside G_(M1) to target Neisseriameningitides.

In other instances, the targeting agent targets liposomes or micellesreversibly conjugated to an siRNA to factors expressed by oncogenes.These can include, but are not limited to, tyrosine kinases(membrane-associated and cytoplasmic forms), such as members of the Srcfamily; serine/threonine kinases, such as Mos; growth factor andreceptors, such as platelet derived growth factor (PDDG), small GTPases(G proteins), including the ras family, cyclin-dependent protein kinases(cdk), members of the myc family members, including c-myc, N-myc, andL-myc, and bcl-2 family members.

In addition, vitamins (both fat soluble and non-fat soluble vitamins)can be used as targeting agents to target biological targets (e.g.,cells) that have receptors for, or otherwise take up, vitamins. Forexample, fat soluble vitamins (such as vitamin D and its analogs,vitamin E, vitamin A), and water soluble vitamins (such as vitamin C)can be used as targeting agents.

Polyethylene Glycol

In some instances, phospholipids of a micelle or liposome describedherein can be modified with or conjugated to polyethylene glycol (PEG).Nonlimiting examples of PEG that can be used in the methods andcompositions described herein include PEGs having a molecular weight ofabout 200 to about 20,000 daltons.

To couple PEG to a phospholipid, the PEG can be activated by preparing aderivative of the PEG having a reactive group at one terminus. Manyactivated derivatives of PEG are known in the art. One nonlimitingexample of an activated PEG derivative is the succinimidyl succinateester of PEG (see, e.g., U.S. Pat. No. 4,179,337). Other nonlimitingexamples of activated PEG molecules that can be used in the methodsdescribed herein include PEGs having a reactive cyanuric chloridemoiety, succinimidyl carbonates of PEG, phenylcarbonates of PEG,imidazolyl formate derivatives of PEG, PEG-carboxymethyl azide,PEG-imidoesters, PEG-vinyl sulfone, active ethyl sulfone derivatives ofPEG, tresylates of PEG, PEG-phenylglyoxal, PEGs activated with analdehyde group, PEG-maleimides, and PEGs with a terminal amino moiety.These PEG derivatives and methods for conjugating such derivatives toagents are known in the art (see, e.g., Zalipsky et al., “Use ofFunctionalized Poly(Ethylene Glycol)s for Modification of Polypeptides”,in Use of Polyethylene Glycol Chemistry. Biotechnical and BiomedicalApplications, J. M. Harris, Ed., Plenum Press, New York (1992); see alsoZalipsky, Adv. Drug Rev. 16:157-182 (1995)).

Therapeutic Administration

The route and/or mode of administration of a liposome or micellereversibly conjugated to an siRNA, described herein, can vary dependingupon the desired results. One with skill in the art, i.e., a physician,is aware that dosage regimens can be adjusted to provide the desiredresponse, e.g., a therapeutic response.

Methods of administration include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, oral, sublingual, intracerebral, intravaginal, transdermal,rectal, by inhalation, or topical, particularly to the ears, nose, eyes,or skin. The mode of administration is left to the discretion of thepractitioner.

In some instances, a liposome or micelle reversibly conjugated to ansiRNA, described herein (e.g., a pharmaceutical formulation of aliposome or a micelle) can effectively cross the blood brain barrier andenter the brain. In other instances, a liposome or micelle can bedelivered using techniques designed to permit or to enhance the abilityof the formulation to cross the blood-brain barrier. Such techniques areknown in the art (e.g., WO 89/10134; Cloughesy et al., J. Neurooncol.26:125-132 (1995); and Begley, J. Pharm. Pharmacol. 48:136-146 (1996)).Components of a formulation can also be modified (e.g., chemically)using methods known in the art to facilitate their entry into the CNS.

For example, in some instances, a liposome or micelle reversiblyconjugated to an siRNA, described herein, is administered locally. Thisis achieved, for example, by local infusion during surgery, topicalapplication (e.g., in a cream or lotion), by injection, by means of acatheter, by means of a suppository or enema, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. In somesituations, a liposome or micelle described herein is introduced intothe central nervous system, circulatory system or gastrointestinal tractby any suitable route, including intraventricular, intrathecalinjection, paraspinal injection, epidural injection, enema, and byinjection adjacent to a peripheral nerve.

Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent, or viaperfusion in a fluorocarbon or synthetic pulmonary surfactant.

A liposome or micelle reversibly conjugated to an siRNA, describedherein, can be formulated as a pharmaceutical composition that includesa suitable amount of a physiologically acceptable excipient (see, e.g.,Remington's Pharmaceutical Sciences pp. 1447-1676 (Gennaro, ed., 19thed. 1995)). Such physiologically acceptable excipients can be, e.g.,liquids, such as water and oils, including those of petroleum, animal,vegetable, or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. The physiologically acceptable excipientscan be saline, gum acacia, gelatin, starch paste, talc, keratin,colloidal silica, urea and the like. In addition, auxiliary,stabilizing, thickening, lubricating, and coloring agents can be used.In one situation, the physiologically acceptable excipients are sterilewhen administered to an animal. The physiologically acceptable excipientshould be stable under the conditions of manufacture and storage andshould be preserved against the contaminating action of microorganisms.Water is a particularly useful excipient when a liposome or micelledescribed herein is administered intravenously. Saline solutions andaqueous dextrose and glycerol solutions can also be employed as liquidexcipients, particularly for injectable solutions. Suitablephysiologically acceptable excipients also include starch, glucose,lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodiumstearate, glycerol monostearate, talc, sodium chloride, dried skim milk,glycerol, propylene, glycol, water, ethanol and the like. Other examplesof suitable physiologically acceptable excipients are described inRemington's Pharmaceutical Sciences (ibid.). The pharmaceuticalcompositions, if desired, can also contain minor amounts of wetting oremulsifying agents, or pH buffering agents.

Liquid carriers can be used in preparing solutions, suspensions,emulsions, syrups, and elixirs containing a liposome or micellereversibly conjugated to an siRNA, described herein. A liposome ormicelle reversibly conjugated to an siRNA, described herein, can besuspended in a pharmaceutically acceptable liquid carrier such as water,an organic solvent, a mixture of both, or pharmaceutically acceptableoils or fat. The liquid carrier can contain other suitablepharmaceutical additives including solubilizers, emulsifiers, buffers,preservatives, sweeteners, flavoring agents, suspending agents,thickening agents, colors, viscosity regulators, stabilizers, orosmo-regulators. Suitable examples of liquid carriers for oral andparenteral administration include water (particular containing additivesdescribed herein, e.g., cellulose derivatives, including sodiumcarboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, e.g., glycols) and their derivatives,and oils (e.g., fractionated coconut oil and arachis oil). Forparenteral administration the carrier can also be an oily ester such asethyl oleate and isopropyl myristate. The liquid carriers can be insterile liquid form for administration. The liquid carrier forpressurized compositions can be halogenated hydrocarbon or otherpharmaceutically acceptable propellant.

In other instances, a liposome or micelle reversibly conjugated to ansiRNA, described herein, is formulated for intravenous administration.Compositions for intravenous administration can comprise a sterileisotonic aqueous buffer. The compositions can also include asolubilizing agent. Compositions for intravenous administration canoptionally include a local anesthetic such as lignocaine to lessen painat the site of the injection. The ingredients can be supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water-free concentrate in a hermetically sealedcontainer such as an ampule or sachette indicating the quantity ofactive agent. Where a liposome or micelle described herein isadministered by infusion, it can be dispensed, for example, with aninfusion bottle containing sterile pharmaceutical grade water or saline.Where a liposome or micelle described herein is administered byinjection, an ampule of sterile water for injection or saline can beprovided so that the ingredients can be mixed prior to administration.

A liposome or micelle reversibly conjugated to an siRNA, describedherein, can be administered rectally or vaginally in the form of aconventional suppository. Suppository formulations can be made usingmethods known to those in the art from traditional materials, includingcocoa butter, with or without the addition of waxes to alter thesuppository's melting point, and glycerin. Water-soluble suppositorybases, such as polyethylene glycols of various molecular weights, canalso be used.

The amount of a liposome or micelle described herein that is effectivefor inhibiting the expression of a target polypeptide, and/or treating adisorder or disease, can be determined using standard laboratory andclinical techniques known to those with skill in the art. In addition,in vitro or in vivo assays can optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed can also dependon the route of administration, the condition, the seriousness of thecondition being treated, as well as various physical factors related tothe individual being treated, and can be decided according to thejudgment of a health-care practitioner. For example, the dose of aliposome or micelle described herein can each range from about 0.001mg/kg to about 250 mg/kg of body weight per day, from about 1 mg/kg toabout 250 mg/kg body weight per day, from about 1 mg/kg to about 50mg/kg body weight per day, or from about 1 mg/kg to about 20 mg/kg ofbody weight per day. Equivalent dosages can be administered over varioustime periods including, but not limited to, about every 2 hours, aboutevery 6 hours, about every 8 hours, about every 12 hours, about every 24hours, about every 36 hours, about every 48 hours, about every 72 hours,about every week, about every two weeks, about every three weeks, aboutevery month, and about every two months. The number and frequency ofdosages corresponding to a completed course of therapy can be determinedaccording to the judgment of a health-care practitioner.

In some instances, a pharmaceutical composition containing a liposome ormicelle reversibly conjugated to an siRNA, described herein, is in unitdosage form, e.g., as a tablet, capsule, powder, solution, suspension,emulsion, granule, or suppository. In such form, the pharmaceuticalcomposition can be sub-divided into unit doses containing appropriatequantities of a liposome or micelle described herein. The unit dosageform can be a packaged pharmaceutical composition, for example, packetedpowders, vials, ampoules, pre-filled syringes or sachets containingliquids. The unit dosage form can be, for example, a capsule or tabletitself, or it can be the appropriate number of any such compositions inpackage form. Such unit dosage form can contain from about 1 mg/kg toabout 250 mg/kg, and can be given in a single dose or in two or moredivided doses.

Kits

A liposome or micelle reversibly conjugated to an siRNA, describedherein, can be provided in a kit. In some instances, the kit includes(a) a container that contains a liposome or micelle reversiblyconjugated to an siRNA, described herein, and, optionally (b)informational material. The informational material can be descriptive,instructional, marketing or other material that relates to the methodsdescribed herein and/or the use of the liposome or micelle, e.g., fortherapeutic benefit.

The informational material of the kits is not limited in its form. Insome instances, the informational material can include information aboutproduction of the liposome or micelle, molecular weight of the liposomeor micelle, concentration, date of expiration, batch or production siteinformation, and so forth. In other situations, the informationalmaterial relates to methods of administering the liposome or micelle,e.g., in a suitable amount, manner, or mode of administration (e.g., adose, dosage form, or mode of administration described herein). Themethod can be a method of treating a subject having a disorder.

In some cases, the informational material, e.g., instructions, isprovided in printed matter, e.g., a printed text, drawing, and/orphotograph, e.g., a label or printed sheet. The informational materialcan also be provided in other formats, such as Braille, computerreadable material, video recording, or audio recording. In otherinstances, the informational material of the kit is contact information,e.g., a physical address, email address, website, or telephone number,where a user of the kit can obtain substantive information about theliposomes or micelles therein and/or their use in the methods describedherein. The informational material can also be provided in anycombination of formats.

In addition to the liposome or micelle, the kit can include otheringredients, such as a solvent or buffer, a stabilizer, or apreservative. The kit can also include other agents, e.g., a second orthird agent, e.g., other therapeutic agents. The components can beprovided in any form, e.g., liquid, dried or lyophilized form. Thecomponents can be substantially pure (although they can be combinedtogether or delivered separate from one another) and/or sterile. Whenthe components are provided in a liquid solution, the liquid solutioncan be an aqueous solution, such as a sterile aqueous solution. When thecomponents are provided as a dried form, reconstitution generally is bythe addition of a suitable solvent. The solvent, e.g., sterile water orbuffer, can optionally be provided in the kit.

The kit can include one or more containers for the liposomes or micellesor other agents. In some cases, the kit contains separate containers,dividers or compartments for the liposomes or micelles and informationalmaterial. For example, the liposomes or micelles can be contained in abottle, vial, or syringe, and the informational material can becontained in a plastic sleeve or packet. In other situations, theseparate elements of the kit are contained within a single, undividedcontainer. For example, the liposomes or micelles can be contained in abottle, vial or syringe that has attached thereto the informationalmaterial in the form of a label. In some cases, the kit can include aplurality (e.g., a pack) of individual containers, each containing oneor more unit dosage forms (e.g., a dosage form described herein) of theliposomes or micelles. The containers can include a unit dosage, e.g., aunit that includes the liposomes or micelles. For example, the kit caninclude a plurality of syringes, ampules, foil packets, blister packs,or medical devices, e.g., each containing a unit dose. The containers ofthe kits can be air tight, waterproof (e.g., impermeable to changes inmoisture or evaporation), and/or light-tight.

The kit can optionally include a device suitable for administration ofthe liposomes or micelles, e.g., a syringe or other suitable deliverydevice. The device can be provided pre-loaded with liposomes ormicelles, e.g., in a unit dose, or can be empty, but suitable forloading.

EXAMPLES

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific substances and procedures described herein. Such equivalentsare intended to be encompassed in the scope of the claims that followthe examples below.

I. Methods

A. Materials

The GFP-siRNA modified with an SPDP group (N-Succinimidyl3-(2-pyridyldithio)-propionate) at the 3′-end of its sense strand [sensestrand: (from 5′ to 3′) AGCUGACCCUGAAGUUCAUTT-SPDP], and theL-glutathione (reduced) were obtained from Invitrogen (Carlsbad, Calif.)and Sigma-Aldrich (St. Louis, Mo.), respectively. The1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (PE-SH, MW 731) and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (PEG-PE) were obtained from Avanti Polar Lipids(Alabaster, Ala.). The d-Salt™ dextran desalting column was obtainedfrom Pierce (Rockford, Ill.). Triethylammonium acetate (TEAA) 1M andchloroform (CH₃Cl) were obtained from Sigma-Aldrich (St. Louis, Mo.) andFisher Scientific (Fair Lawn, N.J.), respectively. The RNase/DNase freewater was obtained from MP Biomedicals (Solon, Ohio) and the phosphatesaline buffer (PBS) 10× solution was obtained from Fisher Scientific(Fair Lawn, N.J.). RNase III E. coli was obtained from Ambion (Austin,Tex.). The thin layer chromatography (TLC) was obtained from EMDChemicals Inc. (Gibbstown, N.J.). Pre-cast 20% TBE gels, SYBR® GoldNucleic Acid Gel Stain, and Lipofectamine™ 2000 Reagent were obtainedfrom Invitrogen.

B. Cell Culture

GFP-expressing C166 endothelial cells were grown in DMEM, at 37° C. and5% CO₂. DMEM and supplements (fetal bovine serum, penicillin,streptomycin and amphotericin B), Trypan Blue Solution and Trypsin wereobtained from CellGro (Kansas City, Mo.).

C. Synthesis of siRNA-S-S-PE Conjugate

A solution of the SPDP-activated siRNA (20 nmoles) in PBS, pH 7.4 (120μl) was added dropwise to a solution of PE-SH (2 μmoles) in DMSO andCHCl₃ (total volume of organic solvents 350 μl). The reaction wascarried out for 48 h at RT with continuous shaking. The unreacted PE-SHwas removed on a desalting column. The collected samples containing thesiRNA-conjugate were ultracentrifuged for 1 min at 14.5×1000 rpm tofurther remove mixed solvents and/or PE-SH. The siRNA-S-S-PE conjugatewas stored at −20° C.

D. Cleavage of Disulfide Bond by TLC

To verify that the disulfide bond in siRNA-S-S-PE conjugate wascleavable in reductive conditions, siRNA-S-S-PE (0.5-1 μg) was incubatedwith 25 μL of a GSH solution (10 mM) in PBS, pH 7.4 (the concentrationof GSH in the cytosol can reach 10 mM). A known amount of the conjugatewas lyophilized and then incubated with the GSH solution at 37° C. for 4h with continuous shaking. The sample was immediately frozen at −80° C.,then lyophilized and finally re-dissolved in water for analysis by TLCusing a mobile phase of CHCl₃:MeOH/8:2. After the TCL was dried, it wasdipped in Molybdenum Blue dye to highlight the presence of a blue spotcorresponding to liberated phospholipid.

E. Preparation of siRNA-S-S-PE/PEG-PE Nanosized Mixed Micelles

A thin polymeric film of PEG-PE was prepared from a chloroform solution(as described in Musacchio et al., Mol. Pharm. 6:468-479 (2009) andSawant et al., Int. J. Pharm. 374:114-118 (2009)). Chloroform wasremoved by N₂, and the film was further dried under vacuum. AsiRNA-conjugate in water was added to the PEG-PE film at weight ratiosof 1:200, 1:500, and 1:750 in a final total volume of 300 μl, and thesystem was extensively vortexed to form nanosized mixed micelles.

F. Micelle Properties

Micelle mean size and size distribution were determined by dynamic lightscattering (DLS) using a Zeta Plus Instrument (Brookhaven InstrumentCo., Holtsville, N.Y.). Size distribution and morphology ofsiRNA-containing mixed micelles were also examined using a transmissionelectron microscopy (TEM). Various samples of PEG-PE andsiRNA-S-S-PE/PEG-PE micelles were diluted to a concentration of 1 mg/mLin deionized water. The micelles were stained with 1% uranyl acetate (asdescribed in Tang et al., J. Natl. Cancer Inst. 99:1004-1015 (2007)),placed on a circular copper grid and examined with a JEOL JEM-1010electron microscope (JEOL USA, Inc, Peabody, Mass.).

Zeta-potential (ζ) of all micelle formulations (diluted in 1 mM KCl) wasmeasured by a Zeta Phase Analysis Light Scattering (PALS) with anultrasensitive zeta potential analyzer instrument (BrookhavenInstruments, Holtsville, N.Y.).

G. siRNA-S-S-PE Conjugate Incorporation into PEG-PE Nanosystem by HPLC

All micelle formulations were analyzed by HPLC (Hitachi, L-7450A)equipped with a UV detector at 260 nm on a XBridge C18 column (Waters,Milford, Mass.). The analyses were carried out at RT with a mobile phaseA composed of 5% acetonitrile in 0.1 M TEAA, pH 7, and mobile phase B of15% of acetonitrile in 0.1 M TEAA. The analyses were run in a gradientelution from 20% to 58% of B in 15 min with a 1 min/ml flow rate. Thesame concentration (1.5 μg/50 μl) of freshly synthesized siRNA-S-S-PEconjugate alone and formulated as different weight ratios of mixedmicelles were compared, and the degree of incorporation was evaluated bynormalizing the area under the peak of free siRNA-conjugate and thesiRNA-conjugate in mixed micelles at the same retention time (t_(r)about 12 mins). As a reference, plain PEG-PE micelles were used.

H. Stability of siRNA Against Degradation by RNase

Freshly prepared siRNA-S-S-PE conjugate was formulated with PEG-PE intoa 1:750 (wt/wt) mixed micelle preparation (a total amount of 4.8 μgsiRNA-S-S-PE) in a final volume of 100 μl. RNAse III E. coli was addedto the formulation and the sample was incubated at 37° C. The stabilityof siRNA in mixed micelles was compared to that of the native siRNA overa 24 h period. At determined time-points, an aliquot of the sample waswithdrawn, frozen in dry ice to stop the progression of degradation andstored at −80° C. until the moment of the analysis. Nucleic aciddegradation was visualized by gel electrophoresis (20% TBE gel). Theanalysis was carried out according to the conditions suggested by theprovider. The bands were stained with SYBR® Gold Nucleic Acid Gel Stainin TBE buffer, and visualized by Kodak MI software.

I. GFP Down-Regulation in C166-GFP Endothelial Cells Estimated by Flow

Cytometry

GFP-C166 endothelial cells were grown in 25 cm² flasks at 37° C. and 5%CO₂ in DMEM containing 10% FBS until 80% confluent. 3.5×10⁴ cells/wellwere seeded in twelve-well plates. After an overnight incubation(confluence about 80%), they were washed once using DMEM (1 mL) andincubated with siRNA-S-S-PE/PEG-PE micelle formulations (each containing84 nM siRNA). For comparisons, the same concentration of naked siRNA andsiRNA in Lipofectamine were used. Fresh micelle samples were incubatedin 300 μl of DMEM with 10% FBS. After 4 h incubation, the medium wasreplaced with 1 ml of fresh DMEM, and kept for a total of 48 h.Untreated cells were used as a control. After the incubation, the cellswere washed once with 200 μl of trypsin solution to remove inactivatingtraces of serum, detached with an additional 200 μl, collected in 15 mltest tubes, and fixed in 1 ml of a 4% paraformaldehyde solution (at 4°C.) until analysis by flow cytometry (BD FACSCalibur). The genesilencing percentage was expressed as the average of three independentexperiments.

J. Cytotoxicity by Trypan Blue Exclusion

The cell viability after the incubation with siRNA micelle formulations,naked siRNA and siRNA in Lipofectamine was tested in the same conditionsused for the GFP-gene silencing test by the Trypan Blue solutionexclusion assay. After incubation with micelles, the cells were washedonce with DMEM and once more with 200 μl of trypsin solution. When thecells were detached from the bottom of the well, a 5-fold volume excessof complete DMEM was added. The cells were recovered and counted inTrypan Blue solution on a known dilution of cell suspension. Cellviability of each formulation was expressed as a percentage of the totalnumber of the treated cells versus the total number of the untreatedcells. The experiment was done in triplicate on three different samplepreparations.

II. Results

A. siRNA-S-S-PE Conjugate

The PE-SH was conjugated to the 3′-end of the modified siRNA sensestrand by introducing a disulfide linkage according the schematic inFIG. 1A. The formation of the siRNA-S-S-PE conjugate was confirmed byits cleavability in a 10 mM GSH solution, mimicking intracellularreductive conditions. Equal amounts of siRNA-S-S-PE conjugate before (C)and after hydrolysis (H), and free PE (P) were run on TLC (FIG. 1B). Twospots were revealed for the sample H, corresponding to the liberatedsiRNA (lower spot) and cleaved phospholipid (upper spot). The startingsiRNA-conjugate showed no impurities before the incubation in the GHSsolution. The free PE-SH and the cleaved phospholipid had differentretention factors compared to each other due to the different dissolvingmedia used (chloroform for the free PE-SH and buffer for the cleavedone) that can affect the run on the TLC.

B. Nanosized Mixed Micelles of siRNA-S-S-PE and PEG-PE

The siRNA-S-S-PE conjugate incorporated readily into PEG-PE micelles viathe PE moiety. The schematic structure of the resulting mixed micelle isshown in FIG. 2A. Both plain PEG-PE micelles and siRNA-S-S-PE/PEG-PEmixed micelles had a mean size around 10 nm by dynamic light scattering(FIG. 2B). Transmission electron microscopy (TEM) analysis demonstratedthe round shape and confirmed their narrow size distribution (FIG. 2C).FIGS. 2B and 2C present the size distribution data for the 1:750 (wt/wt)siRNA-S-S-PE/PEG-PE micelles (similar results were obtained for 1:200and 1:500 mixed micelles).

Zeta potential (ζ) measurements of the mixed micelle and plain PEG-PEmicelle suspensions diluted with 1 mM KCl demonstrated that the surfacecharge of siRNA-containing micelles was more negative (ζ=−28.3±5.6 mV)than that of the plain PEG-PE micelles (ζ=−13.2±3.2 mV) (FIG. 2D).

HPLC analysis demonstrated that the peak at about 2.5 min correspondedto siRNA-S-S-PE-containing mixed micelles, while the peak at about 12min corresponded to the free siRNA conjugate (FIG. 3A). Plain PEG-PEmicelles injected at the same concentration as mixed micelles showed asmall peak at 2.5 min at 260 nm (FIG. 3B). The increase of the area atthis t_(r) for mixed micelles was attributed to the presence of thesiRNA-S-S-PE conjugate in the PEG-PE micelles. Chromatography alsoconfirmed that in the formulations with weight ratios of 1:200 and1:500, siRNA had 62 and 69% incorporation respectively (FIGS. 3C and3D), while at the 1:750 ratio (FIG. 3E) quantitative incorporation ofsiRNA conjugate into the mixed micelles was observed (no free siRNAconjugate peak was seen at 12 min).

C. Stability Against Digestion by RNase

To test the stability of siRNA wrapped into PEG-PE nanoparticles againstenzymatic degradation, siRNA-S-S-PE/PEG-PE mixed micelles were preparedat a 1:750 weight ratio and incubated in the presence of RNase III fromE. coli for 24 h at 37° C. The degradation of siRNA was studied by gelelectrophoresis by following the fluorescence intensity of the siRNAafter SYBR gold gel staining. As shown in FIG. 4B, there were nodegradation products of siRNA in PEG-PE micelles after over 24 h (thesame amount of siRNA not exposed to RNase was used as a positivecontrol). In FIG. 4, the first lane represents the siRNA not digestedand used as reference for both naked siRNA and siRNA-S-S-PE in mixedmicelles; 1=30 mins incubation in RNAse solution, 2=1 h, 3=2 h, 4=3 h,5=4 h, 6=5 h, 7=6 h, and 8=24 h incubation, respectively. The enzymaticdegradation of free (native) siRNA was evident after 30 min (FIG. 4A).

D. GFP-Silencing in GFP-C166 Endothelial Cells

The gene silencing efficiency mediated by the siRNA delivered insidecells as siRNA-S-S-PE/PEG-PE nanosized mixed micelles (1:750 weightratio) was studied using GFP-expressing C166 endothelial cells, in DMEMmedia with 10% serum. Flow cytometry data demonstrated thedown-regulation of the GFP production, confirming the presence of theactive free siRNA inside cells (FIG. 5C). Thus, while the naked siRNAincubated with GFP-C166 cells caused a decrease in GFP production ofonly about 0.5%, the same quantity of siRNA in mixed micelles resultedin almost 30% gene silencing (FIG. 6A).

E. Cell Viability

The comparison of cytotoxicity of siRNA-S-S-PE/PEG-PE mixed micelleswith that of siRNA formulated in the standard transfection reagent,Lipofectamine, which provided a comparable level of the target genesilencing, demonstrated that the cell viability by Trypan Blue exclusionwas close to 100% for all mixed micelle formulations (1:200, 1:500, and1:750 weight ratios), whereas the viability of the cells subjected tothe action of siRNA formulated with Lipofectamine (at the same quantityof siRNA) was less than 20% (FIG. 6A). Thus, the micellar formulationslacked the toxic effect associated with the use of such transfectingagents and observed with electrostatic complex-based systems.

EQUIVALENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

The invention claimed is:
 1. A composition comprising: a micelle or aliposome, the micelle or the liposome comprising: phospholipids; ansiRNA reversibly linked to a first phospholipid, wherein thesiRNA-phospholipid conjugate is part of the layer structure of themicelle or the liposome, wherein the siRNA is linked to the phospholipidby a reversible disulfide bond, and polyethylene glycol (PEG) conjugatedto an additional phospholipid of the micelle or the liposome, whereinweight ratio of the siRNA-phospholipid conjugate to the PEG-phospholipidconjugate is about 1:200 to about 1:750.
 2. The composition of claim 1,wherein the siRNA linked to the phospholipid exhibits reduceddegradation by RNase after the siRNA unconjugates from the phospholipidrelative to siRNA not previously linked to the first phospholipid. 3.The composition of claim 1, wherein the composition further comprises atargeting agent conjugated to the phospholipid of the micelle or theliposome.
 4. The composition of claim 1, wherein the additionalphospholipid in the PEG-phospholipid conjugate is different from thephospholipid in the siRNA-phospholipid conjugate.
 5. The composition ofclaim 1, wherein the additional phospholipid in the PEG-phospholipidconjugate is the same as the phospholipid in the siRNA-phospholipidconjugate.
 6. The composition of claim 1, wherein any one of thephospholipids is phosphatidylethanolamine.
 7. The composition of claim1, wherein the siRNA is linked to the phospholipid at a 3′ end of thesiRNA.
 8. The composition of claim 1, wherein the weight ratio of thesiRNA-phospholipid conjugate to the PEG-phospholipid conjugate is about1:200.
 9. The composition of claim 1, wherein the weight ratio of thesiRNA-phospholipid conjugate to the PEG-phospholipid conjugate is about1:500.
 10. The composition of claim 1, wherein the weight ratio of thesiRNA-phospholipid conjugate to the PEG-phospholipid conjugate is about1:750.